Biotech industries and laboratories are pushed to increase their productivity, efficiency and to lower their costs. Since most of the work involves liquid handling using a broad range of instruments and methods (e.g. pipettes, centrifuges, ovens, vortexes, chromatographic procedures and electrophoretic devices), the full integration of all these steps into a single analytical platform has become a tentative route to low-cost and high-throughput analysis.
The concept of integration by miniaturization of analytical systems stems from the micro-scale electronic industry, which managed to integrate multiple electronic devices into centimeter scale structures to perform a multitude of electrical processes. Such devices are faster, cheaper and easily assembled for industrial scale production. From this field, terms such as microchannels, microfluidics and micropumps emerge as solutions for the integration of different analytical assays and methods into a single format. In addition to high throughput and low costs, miniaturization leads to a reduction of sample, sample loses, and reagent consumption as well as of waste production.
An ideal fully integrated microfluidic device should permit complete analysis, from sample introduction (sample storage and injection), via pre-treatment (sample clean-up, buffer exchange, concentration, mixing, dilution, and derivatization), to the final analysis (e.g. DNA and protein sequence analysis, mass spectrometry), Although a significant progress has been achieved in this field, (e.g. use of electroosmotic flow for sample manipulation and injection, incorporation of Capillary electrophoresis (CE) for DNA, protein and peptide separations), systems are far from performing a broad range of functions. In the following, we examine some important problems that remain to be resolved.
Enzymatic reactions are widespread in clinical diagnosis, industrial applications and academic research. The miniaturization of reactions gives several advantages in relation to conventional schemes, such as faster reactions using less reactants, possibility to perform more assays per unit area and the potential to combine reactions with on-line separations. An important field where microreactors are advantageous in relation to present technologies is in drug discovery. Since the screening for new drugs normally involves a large number of inhibition or activation assays for a particular enzyme, miniaturization of the enzymatic reaction would increase the throughput and decrease the amount of reactants, decreasing the total cost of the assays.
The problems related to incorporation of enzymatic reactions into micrometer scale structures are mainly difficulties to mix two or more solutions (reagent and substrate) and the construction of incubation chambers. One-step reactions can be carried out if the reaction time is not more than a few minutes to keep the diffusion of the analytes along the microchannel at an acceptable level. If it is a multiple step reaction, the situation is more complicated since these often demand removal of the reagent or exchange of the buffer, which leads to mixing problems in nanoliter channels.
Another approach is immobilization of the enzyme to a solid support. As will later be explained, some technological problems arise with such an approach. In addition, another drawback of using solid supports is that the immobilization step cannot be carried out long before the use of the device, due to the possibility of contamination and enzyme degradation, Because of the practical disadvantages and shortcomings of the present technologies, those methods are unlikely to be implemented into fully integrated microfluidic devices.
Sample Pretreatment
The problem to incorporate sample pretreatment into the microchip format is related to the fact that most methods are based on solid stationary phases. The molecules of interest are captured via their affinity to the solid support and once the sample is retained, another solution is employed to wash away salts and other nondesirable compounds which is followed by release of the captured molecules into a buffer suitable for further analysis. Since the molecules are retained to the solid support during sample loading and washing, a significant preconcentration effect is generally obtained. However, there is a problem to incorporate this technology into the micrometer size channel format since the solid support must be mechanically packed using fits, or else be chemically bonded to the wall of the channel. Both approaches are difficult to apply in large-scale production. Another limitation is the specific physical interaction employed to retain the biomolecules of interest. Because of the broad distribution of size, polarity and other molecular properties (e.g. DNA, proteins, peptides), different solid supports are necessary to use in different analytical situations, This is clearly disadvantageous since a different chip would be necessary to manufacture for every new type of component processed. Lastly, fouling of the solid supports due to contaminants and small particulates in real samples limits their use and their capacity for re-use.
Capillary electrophoresis (CE) is a separation method with a high potential for use in the microchip format, which needs the incorporation of sample preparation to reach its full potential. The use of electrophoresis to separate DNA and proteins has been essential for the remarkable achievements of modern biological science. Although conventional slab gel electrophoresis is still very useful and widely used, CE is now the most powerful tool for DNA sequence analysis. In general, the advantages of CE are: higher speed, higher separation efficiency, reduced reagent volumes, and easier automation in relation to the slab gel technique.
The next step is implementation of CE into the chip format. Several parallel channels for electrophoresis can be built within a few centimeters including sample, buffers and waste reservoirs. Actually, Agilent Technologies is marketing a “Labchip” for DNA sizing, RNA analysis and protein separation. The microfabricated device is the simplest automated system for analysis of DNA, RNA and protein in the market. However, the system has no sample preparation capabilities. Desalting and removal of leftover nucleotides and templates is necessary for an adequate injection and separation of DNA. This step is, to some degree, the bottleneck in high-throughput DNA sequencing or analysis and eventually it will be a function necessary to implement onto chip systems. In addition, the use of small-bore capillaries limits the sample volume that can be injected to 1-10 nL and together with the short path-length available for optical detection systems, it is often necessary to include a preconcentration step for proper detection. Since proteins are not amplifyable, as is DNA, a preconcentration step is often needed for analysis by CE.
In summary, for a real analytical integration, the microfluidic approaches must find better methods to perform sample preparation and microreactions. The incorporation of these methods will lead to a significant increase in applicability to many fields, and thus increase the market share. For technological reasons, present technologies do not give a proper solution. The invention described here presents several advantages over the current technologies.